The overall objective of this research is to establish the role of connective tissue in tumor growth and metastasis. It has been shown that collagen synthesis by fibroblasts is transformation sensitive. Since epithelial cell carcinomas are more common in humans, our aim in this grant was to examine collagen synthesis by chemically transformed epithelial cells. Our previous evidence showed that the established rat liver epithelial-like cell line, K16, produced type I collagen. However, the chemically transformed cell line, W8, derived from K16, had lost the ability to synthesize the alpha 2(I) chain. This cell system then became an interesting model system to elucidate the reason for the absent alpha 2(I) chain after chemical transformation. Therefore, we have isolated mRNA from both cell types and established that there is no alpha 2(I) mRNA present in the cytoplasm or the nucleus. Using Southern blot analysis of restricted DNA from both cell types, we have determined that the gene for alpha 2(I) is present and does not contain any large deletions or insertions either in the 3' or promoter 5' region of the gene. Further analysis of gene methylation showed that the 5' promoter region of the W8 alpha 2(I) gene was methylated whereas the corresponding portion of K16 gene was not methylated. Other portions of the alpha 2(I) gene and other genes (tubulin and alpha 1(I) chain of collagen) were partially methylated to a similar extent. The evidence to date suggests that transcription of the W8 alpha 2(I) gene is shut off by methylation in the promoter region of the gene. Further studies are necessary to prove that methylation stopped transcription. (A)